In recent years, with the technical progress in the field of molecular biology, genome structures of various organisms are being revealed. Further, the elucidation of genome structures has revealed that gene mutations participate in various genetic and viral diseases. Accordingly, in various fields of medicine, forensic medicine, molecular biology and the like, the establishment of a method for detecting and analyzing specific gene sequences and mutations in gene sequences has become an important task.
Until now, various methods for detecting and analyzing gene mutations through spectroscopic, biochemical or electrochemical means have been studied. Presently, in general, electrophoresis and high-performance liquid chromatography (HPLC) are applied to detect and analyze gene mutations. However, both of these conventional methods were problematic in that simultaneous analysis of a large number of samples is difficult, that the analysis itself is time-consuming, and that the accuracy is not high.
In recent years, an electrochemical measurement method, which possesses high measuring accuracy and enables detection with trace amounts of reagents as well as simultaneous analysis of many samples, has attracted much interest, and various modified electrodes have been studied and reported as a biosensor (Wang, J., Anal. Chem. 1999, 71, 328).
Specifically, initially, an enzyme electrode comprising an enzyme immobilized on the surface of an electrode as a film, which enables detection by converting changes in substances into electric signals by enzyme reaction, was reported. However, in order to apply this method to the detection and analysis of genes, an enzyme that specifically reacts with the desired analyte (target gene) had to be selected, making its applicability low and problematic.
Hence, recently, DNA biosensors which detect hybridization between probes immobilized on the electrode and target genes as electric signals are being intensely studied. For example, Patolsky et al. have reported immobilizing biotinylated oligonucleotide probes on gold electrodes through phosphothiolate groups, and allowing the probe and the target single-stranded DNA to be in contact, thereby forming a hybrid; further, an enzyme to which avidin that specifically interact with biotin is bonded to is reacted therewith and DNA is detected from the electric signal emitted by the enzyme reaction (Langmuir, 1999, 15, 3703). However, this method requires a large number of procedures such as probe immobilization, hybridization, protein interaction, enzyme reaction and deposition of the product onto electrodes, and thus could not be called a simple method.
Further, Takenaka et al. have reported a method in which DNA is detected by a naphthalene-ferrocene redox intercalator using an oligonucleotide probe chemisorbed onto gold electrodes via a thiol anchor (Anal. Chem. 2000, 72, 1334). However, although it in generally possible to distinguish between single-stranded DNA and double-stranded DNA in methods that use an intercalator as the electrode-active probe, because the binding region of the intercalator is sequence-dependent, the method was problematic in that an intercalator that corresponds to the analyte had to be selected.
Furthermore, De Lumley-Woodyear et al. reported a method in which enzyme (HRP)-labeled (dT)25-30 or (dA)25-30 is covalently attached to a conductive redox polymer film formed on a glassy carbon electrode, and electric signals based on the enzyme reaction caused by its hybridization with complementary oligonucleotides are detected (Anal. Chem. 1999, 71, 394). However, such methods that use enzyme-labeling require intricate procedures when labeling the oligonucleotide with an enzyme, and the excess enzyme has to be removed by cleansing, causing problems in that the measuring accuracy is not necessarily stable.
The present invention has been achieved under the foregoing circumstances, and its object is to provide, upon solving the problems associated with the prior art, a simple, highly applicable method for specifically detecting a nucleic acid with an arbitrary sequence.